In search of a non-surgical alternative for male animal sterilization, this study investigated the use of gold-coated maghemite nanoparticles (γ-Fe₂O₃@Au) functionalized with citrate to produce testicular photohyperthermia (PHT). Wistar rats received an intratesticular injection of the fluid containing the nanoparticles (150 µL/testicle) followed by testicular irradiation with an LED light (808 nm). Testicular temperature was maintained at ~45 °C for 15 min. The results demonstrated a significant reduction in testicular volume and weight and sperm motility and normal morphology in PHT-treated animals, together with histopathological degeneration of seminiferous tubules. No treatment-related side effects or signs of systemic toxicity were observed. The biodistribution of the gold (Au) and iron (Fe) from the nanoparticles showed that the testes were the primary site of nanoparticle accumulation until day 56 post-treatment with possible renal excretion of Au. These findings support the prospect of testicular PHT mediated by γ-Fe₂O₃@Au nanoparticles as a neutering method for male animals.